New purification schemes were developed for isolating large quantities (5 mg) of RNA polymerase I and II enzymes from calf thymus. In addition to the two major fores of RNA polymerase (I, II) previously isolated from calf thymus, a third form of the enzyme was detected and characterized as RNA polymerase III. The enzyme was purified 100,000-fold and exhibited the following properties: alpha-amanitin resistance, elution after polymerase II on DEAE-sephadex chromatography, broad salt optimum (0-0.2M(NH4)2SO4), preference for native versus denatured DNA, and a low Mn/Mg ratio of 1.5. In vitro transcription experiments with SV40DNA (form I) and purified RNA polymerase II enzymes demonstrated that the RNA products were symmetrically transcribed from this template. Dissociation and reconstitution experiments with purified RNA polymerase IIB were performed under wide variety of conditions in which E. coli RNA polymerase (holoenzyme) was reversibly reconstituted. None of the conditions tested resulted in even partial RNA polymerase IIB recovery of activity. Our initial results from these studies and the in vitro transcription experiments might indicate that a specific initiation/assembly factor, like sigma factor in prokaryotic systems, is not an integral part of polymerase IIB as isolated. Alternatively, the polymerase subunit(s) might be modified or proteolytically cleaved after assembly in vivo such as to preclude in vitro reassembly.